Inhibition of fibrosis and AF by TGF-beta inhibition in the posterior left atrium (PLA)

ABSTRACT

The disclosed methods pertain to diagnosing whether a non-ablative, gene therapy is needed for reducing AF fibrosis in a subject, and if so, methods of reducing AF fibrosis in a subject using gene therapy with a dominant negative TGF-β R2 cDNA expression vector. Kits and computer program products are also described, wherein the kits provide materials for diagnosing and treating AF fibrosis, and the computer program products include a computer readable medium having computer readable program code for monitoring the efficacy of therapeutic ablation of fibrosis in a subject using a gene therapy method.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims benefit of priority under 35 U.S.C. 119 to U.S.provisional patent application Ser. No. 61/644,285 filed May 8, 2012,and entitled “INHIBITION OF FIBROSIS AND AF BY TGF-BETA INHIBITION INTHE POSTERIOR LEFT ATRIUM (PLA),” and U.S. provisional patentapplication Ser. No. 61/644,291 filed May 8, 2012, and entitled “USINGINTRACARDIAC ELECTROGRAMS TO PREDICT LOCATION OF FIBROSIS AND AUTONOMICNERVES IN THE HEART,” the contents of which are herein incorporated byreference in their entirety.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

This invention was made with government support under R01HL093490awarded by the National Institutes of Health. The government has certainrights in the invention.

FIELD OF THE INVENTION

The invention relates generally to methods of detecting fibrosis incardiac tissue and using non-ablative, gene therapeutic treatmentmodalities for inhibiting fibrotic tissue development in cardiacdisease.

BACKGROUND

Atrial fibrillation (AF) is a complex arrhythmia with a variety ofunderlying molecular and structural mechanisms contributing to avulnerable AF substrate. The complexity of AF substrate seems to bereflected in the characteristics of AF electrograms (EGMs), with AF EGMmorphology in paroxysmal AF being different than in more persistent AF.However, the precise structural and functional mechanisms that lead tothe formation of AF EGMs have not been well elucidated. The need for abetter understanding of the mechanisms underlying AF EGM formation isheightened by several recent descriptions of regions of high-frequencyactivity during AF called complex fractionated atrial EGMs (CFAEs).Several recent reports suggest that ablation of CFAEs seems to increaseAF ablation success.

In the setting of structural heart disease, specifically heart failure(HF), a variety of mechanisms (for example, changes in ion-channelexpression and gap junction distribution, inflammation, oxidativestress, and a variety of structural changes) are thought to contributeto the creation of a vulnerable AF substrate. Of the structural changesthat occur in the HF atrium, fibrosis is considered to be especiallyimportant in creating conditions conducive to the genesis andmaintenance of AF. In more structurally normal hearts, other mechanisms(for example, heightened autonomic activity) are thought to play a moredominant role in the genesis of AF.

Traditional pharmacological therapies, as well as recently developedsurgical and ablative procedures for AF have had variable success.Surgical and ablative approaches are empiric (anatomic) and do notassess and target patient-specific pathophysiologic derangements. Sincethe ectopic foci that contribute to AF frequently arise in the pulmonaryveins (PVs) and posterior left atrium (PLA), current ablative orsurgical procedures are focused on electrically isolating these regionsfrom the rest of the left atrium. Success rates of these procedures havebeen reported to improve by adding additional ablation or surgicallesions in the atria and by targeting regions demonstrating complexatrial fractionated electrograms (CFAEs), but these are not consistentfindings. Furthermore, this increase in success rates is accompanied byan increase in complications and a decrease in atrialtransport/contractile function.

In view of the limitations of current ablation/surgical approaches,there is a long-felt need to better define the mechanisms underlying AFand to develop novel therapies that specifically target thesemechanisms.

BRIEF SUMMARY

In a first respect, a method of diagnosing whether a non-ablative, genetherapy is needed for reducing AF fibrosis in a subject is disclosed.The method includes three steps. The first step is performing at leastone EGM analysis of a plurality of recorded atrial EGMs for a tissue ina region suspected of having AF fibrosis. The second step is determiningone or more correlations of at least one AF EGM characteristic to aregion having AF fibrosis from the plurality of recorded atrial EGMs forthe tissue. The third step is determining a first outcome of executingstep (b) and a second outcome of executing step (b) for the tissue basedupon the one or more correlations of at least one AF EGM characteristicto a region having AF fibrosis. The first outcome triggers a firstdecision to forego therapy of the analysis region and the second outcometriggers a second decision to perform therapy of the analysis region ofthe tissue.

In a second respect, a method of reducing AF fibrosis in a subject isdisclosed. The method includes four steps. The first step is providingan isolated therapeutic DNA comprising a dominant negative TGF-β R2 cDNAexpression vector that encodes and expresses dominant negative TGF-β R2mRNA and protein in vivo. The second step is administering the isolatedtherapeutic DNA to myocardial tissue of the subject. The third step isassessing fibrosis status of plurality of recorded atrial EGMs for aregion of the myocardial tissue after administration of the therapeuticDNA. The fourth step is determining a first outcome of executing step(b) and a second outcome of executing step (b) for a region based uponthe one or more continued significant changes in EGM characteristicswith administration of the therapeutic DNA. The first outcome triggers afirst decision to forego therapy of the analysis and the second outcometriggers a second decision to perform therapy of the analysis region ofthe tissue.

In a third respect, a method of reducing AF fibrosis in a subject isprovided. The method includes providing an isolated therapeutic DNAcomprising a dominant negative TGF-β R2 cDNA expression vector thatencodes and expresses dominant negative TGF-β R2 mRNA and protein invivo; and administering the isolated therapeutic DNA to myocardialtissue of the subject.

These and other features, objects and advantages of the presentinvention will become better understood from the description thatfollows. In the description, reference is made to the accompanyingdrawings, which form a part hereof and in which there is shown by way ofillustration, not limitation, embodiments of the invention.

BRIEF DESCRIPTION OF THE DRAWINGS

The features, objects and advantages other than those set forth abovewill become more readily apparent when consideration is given to thedetailed description below. Such detailed description makes reference tothe following drawings.

FIG. 1 depicts a flow-diagram for one preferred embodiment illustratinga treatment modality method for targeting fibrosis for treatment basedupon analysis of atrial EGM's.

FIG. 2 depicts a flow-diagram for one preferred embodiment illustratinga treatment modality method for targeting fibrosis using a dominantnegative TGR-β receptor-2 gene therapy based upon analysis of atrialEGM's.

FIG. 3A depicts density and inter-electrode spacing of the low-densityplaques (subpanel (i)) and high-density plaques (subpanel (ii)) used foratrial fibrillation (AF) electrogram (EGM) mapping. LAA indicates leftatrial appendage; PV, pulmonary vein (right).

FIG. 3B illustrates an exemplary EGM map of the entire posterior leftatrium (PLA) section, composed of several individual photo-micrographsat 4× magnification (left) and how the PLA section on the left wasdivided into 4 quadrants; for each quadrant, tissue composition (thatis, % fat vs myocardium vs fibrosis was assessed.

FIG. 3C provides a quantitation of tissue composition for each quadrantshown in FIG. 3B.

FIG. 4A depicts graphical data comparisons of mean dominant frequency(DF) in normal (black bars) vs heart failure (HF) (white bars) atria ofeach atrial fibrillation (AF) measure. Comparisons were made for meanand SD of each atrial fibrillation (AF) measure. Comparisons were madefor the posterior left atrium (PLA), pulmonary vein (PV), and leftatrial appendage (LAA). CHF indicates congestive heart failure. Symbolsabove the bar graphs indicate statistical significance of data.

FIG. 4B depicts graphical data comparisons of standard deviation (SD)dominant frequency (DF) in normal (black bars) vs heart failure (HF)(white bars) atria of each atrial fibrillation (AF) measure. Comparisonswere made for mean and SD of each atrial fibrillation (AF) measure.Comparisons were made for the PLA, PV and LAA. Abbreviations are as setforth in FIG. 4A.

FIG. 4C depicts graphical data comparisons of mean organization index(OI) in normal (black bars) vs heart failure (HF) (white bars) atria ofeach atrial fibrillation (AF) measure. Comparisons were made for meanand SD of each atrial fibrillation (AF) measure. Comparisons were madefor the PLA, PV and LAA. Abbreviations are as set forth in FIG. 4A.

FIG. 4D depicts graphical data comparisons of standard deviation (SD)organization index (OI) in normal (black bars) vs heart failure (HF)(white bars) atria of each atrial fibrillation (AF) measure. Comparisonswere made for the PLA, PV and LAA. Abbreviations are as set forth inFIG. 4A.

FIG. 4E depicts graphical data comparisons of mean fractionationinterval (FI) in normal (black bars) vs heart failure (HF) (white bars)atria of each atrial fibrillation (AF) measure. Comparisons were madefor mean and SD of each atrial fibrillation (AF) measure. Comparisonswere made for the PLA, PV and LAA. Abbreviations are as set forth inFIG. 4A.

FIG. 4F depicts graphical data comparisons of standard deviation (SD)fractionation interval (FI) in normal (black bars) vs heart failure (HF)(white bars) atria of each atrial fibrillation (AF) measure. Comparisonswere made for mean and SD of each atrial fibrillation (AF) measure.Comparisons were made for the PLA, PV and LAA. Abbreviations are as setforth in FIG. 4A.

FIG. 4G depicts graphical data comparisons of mean Shannon entropy(ShEn) in normal (black bars) vs heart failure (HF) (white bars) atriaof each atrial fibrillation (AF) measure. Comparisons were made for meanand SD of each atrial fibrillation (AF) measure. Comparisons were madefor the PLA, PV and LAA. Abbreviations are as set forth in FIG. 4A.

FIG. 4H depicts graphical data comparisons of standard deviation (SD)Shannon entropy (ShEn) in normal (black bars) vs heart failure (HF)(white bars) atria of each atrial fibrillation (AF) measure. Comparisonswere made for mean and SD of each atrial fibrillation (AF) measure.Comparisons were made for the PLA, PV and LAA. Abbreviations are as setforth in FIG. 4A.

FIG. 5A depicts graphical data comparisons of dominant frequency (DF)(subpanel (i)), organization index (OI) (subpanel (ii)), fractionationinterval (FI) (subpanel (iii)) and Shannon entropy (ShEn) (subpanel(iv)) in the heart failure (HF) posterior left atrium (PLA) and leftatrial appendage (LAA). The statistics are indicated above the bargraphs in terms of p value. NS, not significant.

FIG. 5B depicts graphical data comparisons of heterogeneity (SD) of DF(subpanel (i)). OI (subpanel (ii)), FI (subpanel (iii)) and ShEn(subpanel (iv)) in the HF PLA and LAA. Abbreviations are as set forth inFIG. 5A.

FIG. 6A depicts graphical comparisons of the relative percentages offat, fibrosis, and myocardium in the posterior left atrium (PLA) vs leftatrial appendage (LAA) in heart failure (HF) dogs are indicated(subpanel i.) and the heterogeneity of fat, fibrosis, and myocardium inthe PLA and LAA of HF dogs (subpanel ii.). PLA, solid black bars; LLA,solid white bars. The statistics are indicated above the bar graphs interms of p value. NS, not significant.

FIG. 6B illustrates histological comparisons in subpanels i (upperleft)-iv (lower right). wherein the individual sections (magnification×4) from the PLA (subpanels i and iii) and LAA (subpanels ii and iv),respectively, highlighting that (1) there is significantly more fat inthe PLA than the LAA and (2) fibrosis, fat, and myocardium are all moreheterogeneously distributed in the PLA than the LAA. In addition,subpanel (i) demonstrates a typical nerve trunk in the PLA. NS indicatesnonsignificant.

FIG. 7A subpanels show the correlation between % fibrosis in theposterior left atrium (PLA) and dominant frequency (DF) (subpanel (i))and fractionation interval (FI) (subpanel (ii)), respectively. Thestatistics are indicated as insets in terms of r and p values.

FIG. 7B subpanels show the correlation between heterogeneity of fibrosisand heterogeneity of DF (subpanel (i)) and FI (subpanel (ii)),respectively. Abbreviations are as set forth in FIG. 7A.

FIG. 7C shows the correlation between change in Shannon entropy (ΔShEn)with autonomic blockade and % fat in the PLA. Abbreviations are as setforth in FIG. 7A.

FIG. 8A illustrates an exemplary embodiment of a posterior left atrium(PLA; subpanel i.) and left atrial appendage (LAA) section (subpanelii.) from 1 animal. Subpanels iii. and iv. show the correspondingorganization index (OI) of the atrial fibrillation (AF) signals recordedfor each of these regions, respectively. The statistics are indicatedbelow each panel in terms of mean and standard deviation (SD) values.

FIG. 8B illustrates an exemplary embodiment of a PLA (subpanel i.) andLAA section (subpanel ii.) from 1 animal. Subpanels iii. and iv. showthe corresponding dominant frequency (DF) of the AF signals recorded foreach of these regions, respectively. Abbreviations are as set forth inFIG. 8A.

FIG. 9A depicts graphical representation of the effects of autonomicblockade in the posterior left atrium (PLA) and left atrial appendage(LAA) on dominant frequency (DF) (subpanel (i)), organization index (OI)(subpanel (ii)), fractionation interval (FI) (subpanel (iii)), andShannon entropy (ShEn) (subpanel (iv)). The statistics are indicatedabove the bar graphs in terms of p value. NS, not significant.

FIG. 9B illustrates exemplary embodiments of PLA electrograms (EGMs)before (subpanel (i)) and after (subpanel (ii)) double autonomicblockade.

FIG. 9C illustrates an exemplary embodiment of the entire PLA sectionbeing mapped (subpanel i.). The circles highlight areas containingseveral large nerve trunks, indicated by white arrows. Subpanels ii. andiii. show the DF of an atrial fibrillation (AF) episode recorded atbaseline and in the presence of double autonomic blockade, respectively.As shown, autonomic blockade resulted in lower DFs in both the uppercircle (≈8−6 Hz) and in the lower circle (≈9−6 Hz). Subpanel iv. shows amagnified view of a single nerve trunk seen in the lower encircled areain subpanel i. NS indicates nonsignificant.

FIG. 10A depicts the expression vector (pUBc/V5-His/lacZ) used for onepreferred embodiment.

FIG. 10B depicts the one preferred embodiment for expressing thednTGFβPR2 cDNA gene construct as a HA-His₆ tagged fusion gene(pUB6c-HA-TβdnRII).

FIG. 11A depicts the AF duration (sec) for left atria for CHF animalsthat received no injection of exogenously-added dominant negative TGF-βR2 cDNA expression vector (left bar graph) vs. CHF animals that receivedinjection of exogenously-added dominant negative TGF-β R2 cDNAexpression vector (right bar graph).

FIG. 11B depicts the inducibility index (number of AF episodeslasting >30 seconds in duration upon response to induction) for CHFanimals receiving injection into PLA tissues of control plasmid lackingthe dominant negative TGF-β R2 cDNA (right bar graph) orpUB6c-HA-TβdnRII containing the dominant negative TGF-β R2 cDNA (leftbar graph). Asterisk (*) indicates statistically significant differencein the compared results.

FIG. 12 depicts an embodiment where the amount of endogenous TGF-β R2mRNA (“TβRII endogenous”) and dominant negative TGF-β R2 mRNA(“TβdnRII”) present in a representative PLA tissue of a CHF recipientfollowing injection of pUB6c-HA-TβdnRII containing the dominant negativeTGF-β R2 cDNA, as determined quantitative RT-PCT (“qRT-PCR”); the graphillustrates the ratio of mRNA transcript level, normalized against theendogenous TGF-β R2 mRNA transcript level; subpanel (i) indicates thecorresponding transcript copy number of the respective mRNA transcriptsby qRT-PCR.

FIG. 13A depicts Western blots hybridized with anti-HA-TβdnRII antibodyor anti-Cadherin antibody to illustrate expression levels for dominantnegative TGF-β R2-specific protein expression from CHF PLA's transfectedwith either control plasmid lacking the dominant negative TGF-β R2 cDNA(upper left panel) or pUB6c-HA-TβdnRII containing the dominant negativeTGF-β R2 cDNA (upper right panel); the lower panels indicate controlprotein levels reflective of loading the PAGE gels prior to blotting(Anti-Cadherin).

FIG. 13B depicts Western blots hybridized with anti-endogenous TβRIIantibody to illustrate expression levels of endogenous TGF-β R2following introduction into PLA tissues of CHF dogs either controlplasmid lacking the control plasmid lacking the dominant negative TGF-βR2 cDNA (left upper panel) or pUB6c-HA-TβdnRII containing the dominantnegative TGF-β R2 cDNA (upper right panel); loading control proteinblots (GAPDH) are shown in the lower panel.

FIG. 13C depicts graphical results of the relative protein expressionlevels of endogenous TGF-β R2 obtained from PLA tissues from CHF dogsinjected with control plasmid lacking the dominant negative TGF-β R2cDNA (left bar graph) or pUB6c-HA-TβdnRII containing the dominantnegative TGF-β R2 cDNA (right bar graph). The asterisk (*) indicates astatistically significant difference in the data.

FIG. 14 (A-F) depicts immunohistochemistry of three representativesamples in matched pairs of PLA (left panel) and LLA (right panel)tissues isolated from CHF recipients injected with pUB6c-HA-TβdnRIIcontaining the dominant negative TGF-β R2 cDNA into only PLA tissues(indicated pairs taken from animals sacrificed at different timesfollowing treatment).

FIG. 14A depicts immunohistochemistry of a representative sample PLAtissue isolated from an CHF recipient injected with pUB6c-HA-TβdnRIIcontaining the dominant negative TGF-β R2 cDNA into only PLA tissues.

FIG. 14B depicts immunohistochemistry of a representative sample LLAtissue isolated from an CHF recipient injected with pUB6c-HA-TβdnRIIcontaining the dominant negative TGF-β R2 cDNA into only PLA tissues.

FIG. 14C depicts immunohistochemistry of a representative sample PLAtissue isolated from an CHF recipient injected with pUB6c-HA-TβdnRIIcontaining the dominant negative TGF-β R2 cDNA into only PLA tissues.

FIG. 14D depicts immunohistochemistry of a representative sample LLAtissue isolated from an CHF recipient injected with pUB6c-HA-TβdnRIIcontaining the dominant negative TGF-β R2 cDNA into only PLA tissues.

FIG. 14E depicts immunohistochemistry of a representative sample PLAtissue isolated from an CHF recipient injected with pUB6c-HA-TβdnRIIcontaining the dominant negative TGF-β R2 cDNA into only PLA tissues.

FIG. 14F depicts immunohistochemistry of a representative sample LLAtissue isolated from an CHF recipient injected with pUB6c-HA-TβdnRIIcontaining the dominant negative TGF-β R2 cDNA into only PLA tissues.

FIG. 15A illustrates Masson's trichrome histochemistry analysis of PLAtissue section from naïve CHF animal that did not receive a geneinjection. Arrows, blue-staining denote regions of fibrosis.

FIG. 15B illustrates Masson's trichrome histochemistry analysis of PLAtissue section from naïve CHF animal (as in FIG. 15A). Symbols as inFIG. 15A.

FIG. 15C illustrates Masson's trichrome histochemistry analysis of PLAtissue section from CHF recipient injected with plasmid containing thedominant negative TGF-β R2 cDNA. Symbols as in FIG. 15A.

FIG. 15D illustrates Masson's trichrome histochemistry analysis of PLAtissue section from CHF recipient (as in FIG. 15C). Symbols as in FIG.15A. The % fibrosis in CHF recipients containing the dominant negativeTGF-β expression vector was significantly lower in CHF recipientsreceiving the injection of DNA than CHF recipients receiving noinjection of DNA (11.4% vs. 30% fibrosis, respectively; p<0.05).

FIG. 16A depicts Masson's trichrome histochemistry analysis ofrepresentative PLA tissue sections from a CHF recipient injected 24 daysearlier with control plasmid lacking the dominant negative TGF-β R2cDNA. Fibrosis, blue staining; Myocardium, red staining.

FIG. 16B depicts a magnification of the section from FIG. 15Aillustrating the presence of fibrosis. Fibrosis and myocardial tissuestaining as in FIG. 16A.

FIG. 16C depicts Masson's trichrome histochemistry analysis ofrepresentative PLA tissue from a CHF recipient injected 24 days earlierwith plasmid containing the dominant negative TGF-β R2 cDNA. Fibrosisand myocardial tissue staining as in FIG. 16A.

FIG. 16D depicts a magnification of the section from FIG. 15Cillustrating the reduction of fibrosis compared with FIG. 15B. Fibrosisand myocardial tissue staining as in FIG. 16A.

FIG. 16E depicts the graphical results of quantifying the % fibrosisevident in PLA tissue from CHF recipients as illustrated in FIGS. 15Band 15D; control % fibrosis in PLA tissues (left bar graph), % fibrosisin PLA tissues from recipients injected with plasmid containing thedominant negative TGF-β R2 cDNA. Asterisk (*), statistically-significantchange in % fibrosis.

While the present invention is amenable to various modifications andalternative forms, exemplary embodiments thereof are shown by way ofexample in the drawings and are herein described in detail. It should beunderstood, however, that the description of exemplary embodiments isnot intended to limit the invention to the particular forms disclosed,but on the contrary, the intention is to cover all modifications,equivalents and alternatives falling within the spirit and scope of theinvention as defined by the embodiments above and the claims below.Reference should therefore be made to the embodiments and claims hereinfor interpreting the scope of the invention.

DETAILED DESCRIPTION

The methods now will be described more fully hereinafter with referenceto the accompanying drawings, in which some, but not all permutationsand variations of embodiments of the invention are shown. Indeed, theinvention may be embodied in many different forms and should not beconstrued as limited to the embodiments set forth herein. Theseembodiments are provided in sufficient written detail to describe andenable one skilled in the art to make and use the invention, along withdisclosure of the best mode for practicing the invention, as defined bythe claims and equivalents thereof.

Likewise, many modifications and other embodiments of the methodsdescribed herein will come to mind to one of skill in the art to whichthe invention pertains having the benefit of the teachings presented inthe foregoing descriptions and the associated drawings. Therefore, it isto be understood that the invention is not to be limited to the specificembodiments disclosed and that modifications and other embodiments areintended to be included within the scope of the appended claims.Although specific terms are employed herein, they are used in a genericand descriptive sense only and not for purposes of limitation.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of skill in the artto which the invention pertains. Although any methods and materialssimilar to or equivalent to those described herein can be used in thepractice or testing of the present invention, the preferred methods andmaterials are described herein.

Moreover, reference to an element by the indefinite article “a” or “an”does not exclude the possibility that more than one element is present,unless the context clearly requires that there be one and only oneelement. The indefinite article “a” or “an” thus usually means “at leastone.”

As used herein, “about” means within a statistically meaningful range ofa value or values such as a stated concentration, length, molecularweight, pH, sequence identity, time frame, temperature or volume. Such avalue or range can be within an order of magnitude, typically within20%, more typically within 10%, and even more typically within 5% of agiven value or range. The allowable variation encompassed by “about”will depend upon the particular system under study, and can be readilyappreciated by one of skill in the art.

Robust clinical diagnostic methods are disclosed that enable use ofAtrial fibrillation (AF) electrograms (EGMs) to detect specific AFmechanisms pertaining to fibrosis and the use of a novel, dominantnegative TGF-β R2 gene expression vector to therapeutically reduce thepercentage fibrosis present in PLA tissues with high selectivity andefficiency. The combination diagnostic-treatment method provides a novelapproach for diagnosing and treating fibrosis in AF conditions.

Contribution of Fibrosis to AF EGMs

The correlation between AF EGM characteristics and the underlyingquantity and distribution of fibrosis was systematically assessed. Usinga variety of time and frequency domain measures, the signalcharacteristics of AF EGMs in the setting of HF (where fibrosis is knownto be a key contributor to the genesis and maintenance of AF) andcompared these with AF EGMs in normal hearts (where AF was induced byvagal stimulation) were examined. The relationship between thecharacteristics of AF EGMs in HF and the underlying distribution ofmyocardium, fibrosis, fat, and autonomic ganglia in the failing leftatrium was also systematically assessed.

Though the details are presented in the Examples, the findings can besummarized as follows:

(1) AF EGM measures are significantly different in AF in the normalversus the HF atrium, with AF being slower (lower DF), more organized(higher OI), and having a higher FI in the setting of HF;

(2) there are significant regional differences in AF signal content inHF, with AF being less organized (lower OI) in the PLA than in the LAA;moreover, all EGM measures are significantly more spatiallyheterogeneous in the PLA than in the LAA;

(3) there is a significantly greater amount of fat in the PLA comparedwith the LAA, with the fat being richly innervated with large nervetrunks; moreover, fibrofatty tissue is more heterogeneously distributedin the PLA than in the LAA; and

(4) AF signal content in the HF atrium correlates with the total amountof fibrosis, with increasing fibrosis correlating with slowing andincreased organization of AF EGMs; furthermore, heterogeneity of AFsignal content in the HF atrium correlates with the heterogeneity ofunderlying fibrosis.

AF EGMs were systematically characterized in 2 well-characterizedsubstrates for AF: (1) in a normal heart where vagal stimulation (withresulting refractory period shortening) is the primary mechanism for AFand (2) in pacing-induced HF where fibrosis is thought to be a dominantmechanism underlying AF, with other mechanisms, such as oxidative stressand autonomic dysfunction, also contributing at least partially to AFsubstrate. AF EGM content was significantly different in normal versusHF atria, with EGMs in HF being significantly slower, and surprisingly,more organized and less fractionated compared with AF EGMs in normalhearts. The strong correlation between the amount and heterogeneity offibrosis and the time and frequency domain measures of AF EGMs in HFreveals that fibrosis contributes to AF EGM characteristics. Patientswith AF, worsening structural heart disease appears to contribute notonly to the increasing chronicity of AF but also to AF EGM content.

EGM differences between HF and normal hearts can provide valuableinsight into the patho-physiologic mechanisms underlying AF and may beof potential clinical significance in patients with AF undergoing AFablation. It is well known that success rates of ablation proceduresdecrease in patients with permanent AF (compared with paroxysmal AF), atleast, in part, because of the presence of structural heart disease inthese patients.

The addition of EGM-guided ablation (for example, CFAE ablation)increases long-term success of these procedures. The increasedregularity of EGMs (indicated by increased OI in HF) in the presence ofslower activation rates (indicated by lower DFs and higher FIs) in HFindicates the presence of regions of underlying fibrosis. An enhancedability to identify islands dense fibrosis (by real-time AF EGManalysis) allows for a greater precision in the placement of linearablation lesions in the atrium.

Thus, one embodiment concerns a method of targeting fibrosis forablation in a subject. The method includes recording an Atrial EGM froma subject (FIG. 1; 1.1). The subject is preferably a mammal; mostpreferably, the subject is a human. The subject is preferably a patientin need of monitoring cardiovascular disease; more preferably, thesubject is a patient in need of preventative treatments for stroke orcongestive heart failure, in particular, where such conditions areattributed to atrial fibrillation (AF); most preferably, the subject isa patient in need of monitoring sustained arrhythmia, such as atrialfibrillation (AF).

The Atrial EGM is preferably recorded in AF or in sinus rhythm. Therecording is preferably obtained by standard procedures well known inthe art.

Once obtained, an analysis of the EGM is performed using one or moreanalytical subroutines (FIG. 1; 1.2). Preferred analytical subroutinesinclude at least one member selected from the group consisting ofdominant frequency analysis (DF), organizational index analysis (OI),fractional interval analysis (FI) and Shannon Entropy analysis (ShEn).The analysis of the EGM can be done on-line (for example, in real-time)or off-line (for example, using previously acquired EGM data provided ina readable computer media).

The analysis of the EGM performed in accordance with one or more of theaforementioned analytical subroutines permits identification of one ormore correlations of at least one AF EGM characteristic to a regionhaving fibrosis. More preferably, one or more aforementioned analyticalsubroutines permits identification of one or more correlations of atleast two AF EGM characteristics to a region having fibrosis. Mostpreferably, one or more aforementioned analytical subroutines permitsidentification of one or more correlations of three AF EGMcharacteristics to a regions having fibrosis.

Preferred correlations include one or more of the following: (i) mean DFnegatively correlates with % fibrosis; heterogeneity of DF negativelycorrelates with heterogeneity of fibrosis; mean FI positively correlateswith % fibrosis; and heterogeneity of FI positively correlates withheterogeneity of fibrosis. Preferably, two correlations are selectedfrom the group of (a) one correlation based upon % fibrosis and (b) onecorrelation based upon heterogeneity of fibrosis.

Thus, determining one or more correlations of at least one AF EGMcharacteristic to a region having fibrosis from the plurality ofrecorded atrial EGMs for the tissue permits the identification of one ormore correlations of an AF EGM characteristic with a region suspected ofhaving fibrosis enables one to connect regions of fibrosis with ablationlesions (FIG. 1; 1.3).

The decision is then made to forego therapy or to perform therapy on agiven AF substrate based upon whether an outcome of the analysis regionsuspected of having fibrosis (FIG. 1; 1.4). If a first outcome of theanalysis indicates that the region contains no evidence of fibrosis(FIG. 1. “NO” at 1.4), then the first outcome triggers a first decisionto forego therapy of the analysis region (FIG. 1; 1.5). If a secondoutcome of the analysis indicates that the region contains evidence offibrosis (FIG. 1, “YES” at 1.4), then the second outcome triggers asecond decision to perform therapy of the analysis region (FIG. 1; 1.6).

Use of Dominant Negative TGF-β R2 Therapy to Improve AF Performance andReduce AF Fibrosis

The aforementioned Atrial EGM analysis of fibrosis tissues in AF permitsidentification of sites within AF tissue for non-ablative therapy. Inparticular, the use of direct injection of isolated expression vectorsencoding dominant negative TGF-β R2 genes into myocardial tissue havingfibrosis provided a dramatic reduction in endogenous TGF-β R2 proteinexpression; improved AF characteristics, both in terms of reduced AFepisode duration and reduced frequency of numerous, prolonged AFepisodes (that is, AF events lasting longer than 30 seconds) uponinduction; and resulted in significant reductions in the percentage offibrosis in the injected tissues (PLA). These details are presented inthe Examples.

Thus, another embodiment concerns a method of improving AF performanceand reducing AF fibrosis in a subject through the selective delivery ofa dominant negative TGF-β R2 cDNA gene to a targeted site withinmyocardial tissue. The method includes providing a dominant negativeTGF-β R2 cDNA expression vector to a subject (FIG. 2; 2.1). Anyexpression vector amenable for gene therapeutic use in a subject is asuitable vector for expressing the dominant negative TGF-β genecassette. The advantages of the selected approach is the preferable useof isolated, non-viral, expression vectors, thereby reducing anypossibility of viral recombination or reversion occurring to formnon-attenuated or live virus that would present possible risk to thesubject.

The subject is preferably a mammal; most preferably, the subject is ahuman. The subject is preferably a patient in need of monitoringcardiovascular disease; more preferably, the subject is a patient inneed of preventative treatments for stroke or congestive heart failure,particularly where such disease conditions are attributed to autonomicnerve tissue activity; most preferably, the subject is a patient in needof monitoring sustained arrhythmia, such as atrial fibrillation (AF).

Once provided, the DNA is introduced into a myocardial tissue (FIG. 2;2.2). Preferred myocardial tissue include those that contain evidence offibrosis. A variety of administration routes for delivering nucleicacids to specific cell types and tissue systems are well known in theart and also fall within the scope of practicing the present method. Apreferred mode of administering isolated DNA to a particular tissue ororgan is by direct injection at the precise site of treatment (forexample, PLA tissue having evidence of fibrosis), simply because thecandidate sites will be identified with the companion diagnostic methodbased upon EGM analysis of AF.

The efficacy of the therapeutic DNA can be monitored followingadministration. Preferably, one can apply the method disclosed herein toexamine the absence or presence of fibrosis recurrence at sites of DNAinjection using the EGM analytical algorithms to assess fibrosis status(FIG. 2, 2.3). Analysis of the EGM performed in accordance with one ormore of the aforementioned analytical subroutines permits assessment ofat least one AF EGM characteristic after administration of thetherapeutic DNA.

Optionally, the efficacy of the therapy also can be monitored byexamining AF performance characteristics. For example, one can monitorAF duration or frequency of AF episodes upon induction (“AFinducibility”).

The decision is then made to forego therapy or to continue therapy on agiven AF substrate based upon whether an outcome of the analysis regionincludes an EGM having a continued significant change in at least oneEGM characteristic in response to administration of the therapeutic DNA(FIG. 2; 2.4). If a first outcome of the analysis indicates that theregion does not contain a continued significant change in at least oneEGM characteristic with administration of the therapeutic DNA (FIG. 2,“NO” at 2.5), then the first outcome triggers a first decision tocontinue the therapeutic approach (FIG. 2; 2.5). For example, a regiondoes not contain a continued significant change is one does not retainor maintain a reduction in % fibrosis as a result of gene therapy; thatis, a previously noted reduction in fibrosis is no longer evident andthat an elevated or increased % fibrosis is now evident. For example,where evidence is obtained that demonstrates that the injectedtherapeutic DNA displays lower efficacy over time (as revealed byincreased % fibrosis), another dose of the therapeutic DNA can beinjected into the subject. Preferably, the selection of the site forinjection can be the same as the previous injection. More preferably,the site of injection is determined anew through use of the EGManalytical algorithms disclosed above (see FIG. 1). The first decision2.5 to continue the therapeutic approach is preferably to repeat theadministration by injection of the TGFβdnR2 cDNA expression vector intomyocardial tissue (FIG. 2, 2.2).

If a second outcome of the analysis indicates that the region contains acontinued significant change in at least one EGM characteristic withadministration of the therapeutic DNA (for example, a retention ormaintenance of reduction in % fibrosis) (FIG. 2, “YES” at 2.4), then thesecond outcome triggers a second decision to forego continued therapy(FIG. 2; 2.6). The second decision 2.6 optionally can direct one tocontinue monitoring fibrosis status by analyzing EGMs (FIG. 2. 2.3) as ameans to monitor efficacy of the therapeutic DNA.

Accordingly, some embodiments according to some aspects of the presentinvention may be realized in hardware, software, or a combination ofhardware and software. Some aspects of some embodiments of the presentinvention may be realized in a centralized fashion in at least onecomputer system, or in a distributed fashion where different elementsare spread across several interconnected computer systems. Any kind ofcomputer system or other apparatus adapted for carrying out the methodsdescribed herein is suited. A typical combination of hardware andsoftware may be a general-purpose computer system with a computerprogram that, when being loaded and executed, controls the computersystem such that it carries out the methods described herein.

Some embodiments according to some aspects of the present invention mayalso be embedded in a computer program product, which comprises all thefeatures enabling the implementation of the methods described herein,and which when loaded in a computer system is able to carry out thesemethods. Computer program in the present context means any expression,in any language, code or notation, of a set of instructions intended tocause a system having an information processing capability to perform aparticular function either directly or after either or both of thefollowing: a) conversion to another language, code or notation; b)reproduction in a different material form.

Some embodiments according to some aspects of the present inventioncontemplate one or more processors operatively coupled to one or morememories (for example, a non-transitory computer readable medium) inwhich one or more steps described herein are stored as instructions orexecutable code in the one or more memories and are executed by the oneor more processors or are used to configure the one or more processors.Some embodiments according to some aspects of the present inventioncontemplate that the one or more processors and the one or more memoriesare part of a computer system. The computer system may be part of, forexample, laboratory equipment or medical equipment.

Some embodiments according to some aspects of the present inventioncontemplate that the one or more processors and/or the one or morememories are part of an integrated circuit and/or an applicationspecific integrated circuit (ASIC) and/or a single integrated circuitchip.

Some embodiments according to some aspects of the present inventioncontemplate using software, hardware and/or firmware.

Some embodiments according to some aspects of the present inventioncontemplate using a software algorithm that can be installed incommercially available EGM imaging and computer machine language-basedanalysis stations The software algorithm may compute, for example,correlation functions obtained from one or more of DF, OI, FI, and ShEnanalyses of EGM recordings to obtain a decision whether to performablation therapy on a selected region. The software algorithm can berealized, for example, in Matlab, C, C++, Pascal, Java, Fortran, Perl,Basic, machine language or other programming languages. To any extent towhich specific processing hardware is provided to realize the algorithm,some embodiments according to some aspects of the present inventionprovide for digital signal processors and/or field programmable gatearray, etc. Some embodiments according to some aspects of the presentinvention also contemplate that a data interface with an existing EGMrecording system provide raw data, which includes spectral data directlyoutput from a recorder.

For example, a computer program product that includes a computerreadable medium having computer readable program code for monitoring theefficacy of therapeutic ablation of fibrosis in a subject using a genetherapy method is provided. The monitoring the efficacy of therapeuticablation of fibrosis includes two steps. The first step is performing atleast one EGM analysis of a plurality of recorded atrial EGMs for atissue treated previously with the gene therapy method. The second stepis executing at least one of the following sets of instructions: (i)determining one or more correlations of at least one AF EGMcharacteristic to a region having previously identified fibrosis fromthe plurality of recorded atrial EGMs for the tissue; (ii) determining afirst outcome of executing step (i) and a second outcome of executingstep (i) for a region based upon the one or more significant changes inEGM characteristics following administration of the gene therapy. Thefirst outcome triggers a first decision that the gene therapy of theanalysis region is efficacious and the second outcome triggers a seconddecision that the gene therapy of the analysis region is notefficacious.

In one aspect, the aforementioned computer program product includes aspart of the first step at least one analytical subroutine selected fromthe group consisting of dominant frequency analysis (DF), organizationalindex analysis (OI), fractional interval analysis (FI) and ShannonEntropy analysis (ShEn). In another aspect, regarding targeting fibrosisfor ablation, the one or more correlations of at least one AF EGMcharacteristic to a region suspected of having fibrosis comprises atleast one correlation selected from the group consisting of (i) mean DFnegatively correlates with % fibrosis; (ii) heterogeneity of DFnegatively correlates with heterogeneity of fibrosis; (iii) mean FIpositively correlates with % fibrosis; (iv) heterogeneity of FIpositively correlates with heterogeneity of fibrosis and combinationsthereof.

Kits are contemplated with the scope of the present disclosure.Preferred components of kits include algorithm-encoded software on acomputer machine readable medium that permits execution of instructionsby a machine for implementing the methods of the present invention toguide in the selection of AF substrate for substrate-guided ablation forHF. Kits can also include other items, such as instructions, manuals,and on-line help sections for assisting users with implementing theexecutable software code. Kits can also include other items, such astherapeutic DNA materials, buffers, reagents and the like.

EXAMPLES

The invention will be more fully understood upon consideration of thefollowing non-limiting examples, which are offered for purposes ofillustration, not limitation.

Example 1 Dog Protocols

Purpose-bred hound dogs (weight range, 25-35 kg) were used in thepresent study for both control and HF groups. This protocol conforms tothe Guide for the Care and Use of Laboratory Animals published by the USNational Institutes of Health (Publication No. 85-23, revised 1996) andwas approved by the Animal Care and Use Committee of NorthwesternUniversity. Before undergoing the procedures listed below, all animalswere premedicated with acepromazine (0.01-0.02 mg/kg) and were inducedwith propofol (3-7 mg/kg). All experiments were performed under generalanesthesia (inhaled) with isoflurane (1%-3%). The adequacy of anesthesiawas assessed by toe pinch and palpebral reflex

Example 2 Canine HF Model

In 21 dogs, HF was induced by 3 to 4 weeks of right ventriculartachypacing (240 beats per minute) by an implanted pacemaker. In 19dogs, a transvenous pacemaker was placed via a jugular approach, underaseptic conditions. In 2 dogs, a pacemaker was placed on the ventriclevia an epicardial approach (that is, via a left lateral thoracotomy).Left ventricular function was assessed during pacing by serialechocardiograms (data not shown). HF was confirmed after 3 to 4 weeks ofpacing. Twenty dogs without rapid ventricular pacing were used ascontrols.

Example 3 Open-chest Mapping

At the terminal study, a left lateral thoracotomy was performed. Lowdensity and high density mapping protocols were used. The low densitymapping protocol was used to compare AF EGMs from 14 HF dogs with EGMsfrom 20 control dogs with AF induced during vagal stimulation. With lowdensity mapping, the posterior left atrium (PLA), left superiorpulmonary vein (PV), and left atrial appendage could be mappedsimultaneously. The PLA and LAA were mapped using two rectangularplaques containing 21 electrodes each (7×3 electrodes, inter-electrodedistance=5 mm) from which 18 bipolar EGMs were recorded. The PV wasmapped with a 40-electrode, rectangular plaque (8×5 electrodes,inter-electrode distance=2.5 mm) from which 35 bipolar EGMs wereobtained. FIG. 3A shows the schematics of the plaques. The signals fromthe low density plaques were recorded and stored at a 977 Hz samplingrate with the GE Prucka Cardiolab system (GE Healthcare, Waukesha,Wis.).

High density mapping was performed in 8 HF dogs for detailed comparisonsof EGMs with the underlying tissue structure. Mapping was performedsequentially in the PLA and LAA with a triangular plaque containing 130electrodes (inter-electrode distance of 2.5 mm) from which 117 bipolarEGMs were recorded. The schematic is shown in FIG. 3A. The UNEMAPmapping system (Univ. of Auckland, Auckland, New Zealand) was used forrecording and storing the EGMs at a 1 kHz sampling rate. Even though wedid not separately map the PVs during high-density mapping (owing to therelatively large surface area of the high density plaques, it wastechnically challenging to cover the PVs, which have a circular anduneven surface), the high-density plaque did straddle the proximal PVsduring PLA mapping. One dog underwent both low and high density mapping.

Example 4 AF Induction

AF was induced in the control animals in the presence of left cervicalvagal stimulation via programmed stimulation (eight S1 beats at 400 msfollowed by a single extrastimulus). For vagal stimulation, the leftcervical vagus nerve was isolated and a bipolar stainless steelelectrode was attached to the nerve. Vagal stimulation was performedusing a Grass S44G stimulator (Astromed, West Warwick, R.I.) with a 5-10V amplitude, a 20 Hz stimulation rate, and a 5 ms pulse width; anadequate vagal response was adjudged by: 1) sinus node slowing by atleast 25% or 2) PR prolongation by more than 25% or 2:1 AV block. AF wasinduced in the HF dogs with burst pacing under baseline conditions usingcycle lengths of 180 ms to 110 ms with 10 ms decrements for 10 secondsfor each cycle length. Current was set at four times threshold forcapture. In 6 of the 8 HF dogs that underwent high density mapping, AFwas also induced in the presence of double autonomic blockade (0.2 mg/kgpropranolol and 0.04 mg/kg atropine), in order to test the hypothesisthat autonomic nerves in the ganglion-rich fibrofatty tissue also affectEGM characteristics. Ten seconds of AF in the middle of a sustainedepisode were recorded when high density mapping was performed, whereasthe entire AF episode beginning at AF initiation was recorded when lowdensity mapping was performed.

Example 5 Histology

The histologic analysis described below (for example, comparison oftissue make-up between PLA and LAA) and EGM-tissue analysis was onlyperformed for HF atria, as these atria are known to harbor significantfibrosis. Normal atria on the other hand are not known to havesignificant fibrosis. In examples of histology from the PLA and LAA oftwo normal dogs, there was significantly less fibrosis in normal heartsas compared to HF hearts (data not shown).

Tissue Sample Preparation.

In the animals undergoing high density mapping, immediately followingthe in vivo electrophysiological study, the heart was promptly excisedout of the chest and immersed in ice-cold cardioplegia as previouslydescribed by us. After marking the exact orientation of the high densityplaques, tissue samples were taken from the PLA and LAA regions of theleft atrium and snap frozen in liquid nitrogen. Samples were saved inthe exact orientation in which high density mapping had been performed.All samples were initially saved at −80° C. The oriented tissue sampleswere frozen in Tissue-Tek OCT (Optimal Cutting Temperature) compound at−80° C.

For paraffinization, the tissue was thawed and a quick wash given toclean off all the OCT. Using a PCF LEICA 1050 Tissue Processor, thetissue was embedded in paraffin. The tissue processor uses 10% NBF(Neutral Buffered Formalin) for fixing and the tissue dehydration isperformed with incremental concentrations of Ethanol (ETOH). ETOH isexchanged with xylene and finally xylene is exchanged with paraffin at58° C. Then tissue is embedded in a paraffin block.

Masson's Trichrome Staining.

Tissue sections were cut 4 μm apart. Paraffin was removed by placing thetissue section in histology grade xylene for two minutes and the processwas repeated four times changing xylene solution after every twominutes. Finally, the xylene was washed away with ETOH for one minute inabsolute ETOH, then again for one more minute with fresh absolute ETOH,followed by wash in 95% ETOH for 30 seconds, and subsequently in 70%ETOH for 45 seconds. ETOH was then washed with water for one minute. Thetissue section was then ready for staining. The section was treated withBouin's mordant at room temperature overnight. The following day thetissue section was rinsed in running water to remove excess yellow. Thetissue section was stained in Weigert's Solution for 7 minutes. Next, itwas dipped once in 1% acid alcohol and immediately rinsed. The sectionwas then stained in Beibrich Scarlet-Acid fuchsin for 2 minutes,followed by a rinse in distilled water. Subsequently, the tissue sectionwas stained in phosphomolybdic-phosphotungstic acid solution for 6minutes, followed by another rinse in distilled water. The issue sectionwas then stained in Aniline Blue solution for 5 minutes, followed byanother rinse in distilled water. Immediately, the tissue was dippedonce in 1% Glacial acetic acid and quickly rinsed. The tissue sectionwas then dehydrated in twice in each concentration of 95% and 100% ofETOH, which was later exchanged with xylene. A coverslip was finallyplaced on the tissue section for microscope examination.

Example 6 EGM Analysis

Custom analysis tools developed in MATLAB (Mathwork, Natick, Mass.) wereused for all offline EGM analysis. The signals were divided into 4second segments to account for any variability of the both the signalsand the measurements of the signals. We have previously shown thatdominant frequencies averaged from multiple 4-second segments were abetter reflection of activation rates than single segments of anylength. The following four measurements were computed.

Dominant Frequency (DF).

DF is a frequency domain measure of activation rate. Following bandpassfiltering with cutoff frequencies of 40 and 250 Hz and rectification,the power spectrum of the EGM segment was computed using the fastFourier transform. The frequency with the highest power in the powerspectrum was considered the DF.

Organization Index (OI).

OI is a frequency domain measure of temporal organization or regularity.It has been shown that AF episodes with recordings with high OI are moreeasily terminated with burst pacing and defibrillation. OI wascalculated as the area under 1-Hz windows of the DF peak and the nextthree harmonic peaks divided be the total area of the spectrum from 3 Hzup to the fifth harmonic peak.

Fractionation Interval (FI).

FI is the mean interval between deflections detected in the EGM segment.Deflections were detected if they meet the following conditions: 1) thepeak-to-peak amplitude was greater than a user determined noise level,2) the positive peak was within 10 ms of the negative peak, and 3) thedeflection was not within 50 ms of another deflection. The noise levelwas determined by selecting the amplitude level that would avoiddetection of noise-related deflections in the iso-electric portions ofthe signal. FIs≦120 ms have been considered CFAE. The 120 ms criterionwas used to calculate the % CFAE in each region for both low density andhigh density mapping. FI is dependent on both the AF cycle length andthe fractionation of the EGM.

Shannon's Entropy (ShEn).

ShEn is a statistical measure of complexity. The 4000 or 3908 (dependingon the 1 kHz or 977 Hz sample rate) amplitude values of each EGM segmentwere binned into one of 29 bins with width of 0.125 standard deviations.ShEn was then calculated in accordance for equation (1).

$\begin{matrix}{{ShEn} = \frac{- {\sum\limits_{i = 1}^{29}\;{p_{i}\log_{10}p_{i}}}}{\log_{10}p_{i}}} & (1)\end{matrix}$

In this equation, p_(i) is the probability of an amplitude valueoccurring in bin i. The above measures were assessed for eachpixel/electrode on each plaque. There was a small number of electrodes(<10%) where signal (EGM) quality was inadequate (for example, due tonoise, poor contact) for assessment of the above measures. These pixelsare shown as grey in FIGS. 8 and 9.

Example 7 Tissue Analysis

Tissue sections were examined at 4× magnification (bright-field). Eachslide was divided into 48 to 110 microscopic fields, depending on thesize of the section (see FIG. 3B, as well as FIGS. 8 and 9; the figuresshow examples of how each slide (section) was divided into multiplecomponent microscopic fields). Digital pictures of these fields weretaken. Digital images were manually edited to remove all tissue elementsthat could not be classified as myocardium, fibrosis, or fat (forexample, blood vessels, nerves, etc.). A custom MATLAB program was usedto semi-automatically classify all pixels in the edited images. In each4× tissue section, myocardium (red), fibrosis (blue) and fat (white)were classified based on the pixels' RGB values. The percentagebreakdown of fibrosis vs. myocardium vs. fat was then calculated foreach 4× tissue section. Mean percentage of fibrosis vs. myocardium vs.fat for an entire PLA or LAA section was taken as the mean of allrespective percentages for each individual 4× section. Heterogeneity offat vs. myocardium vs. fibrosis for a PLA or LAA was calculated as thestandard deviation (SD) of the pixel counts of all the individual 4×sections that comprised that PLA or LAA.

Example 8 Tissue and EGM Correlation

Each tissue section was divided into 4 quadrants. The high-densityrecordings, after being aligned to underlying tissue orientation, werealso divided into 4 quadrants (see schematic in FIG. 3B). In eachquadrant, the absolute amount of fat, fibrosis, and myocardium wasassessed. Linear regression analysis was performed to assess thecorrelation between tissue and EGM characteristics.

Example 9 Statistical Methods

All data are reported as mean±SE. Mixed effects ANOVA was used tocompare the mean DF, OI, FI, and ShEn between HF and normal dogs andamong pulmonary vein (PV), PLA, and LAA. SDs to quantify spatialheterogeneity were also analyzed in a similar fashion. In the HF dogsthat underwent high-density mapping, comparison of EGMs between the PLAand LAA were made using unpaired t tests (as these regions were mappedat separate times during the electrophysiological study [that is, notsimultaneously as was the case with low-density mapping]). Comparisonsof tissue characteristics between the PLA and LAA were made using pairedt tests. Before and after comparisons made in the same animals (forexample, before and after double autonomic blockade) were assessed forsignificant differences via paired t tests.

Tissue and EGM correlations were performed by dividing each tissuesection into 4 quadrants paired with the EGM characteristics (DF, OI,FI, and ShEn) of the high-density maps similarly divided into 4quadrants and performing linear regression analysis. P≦0.05 was taken assignificant for all the above analyses.

Example 10 Canine Congestive Heart Failure (CHF) Model

Purpose-bred hound dogs were used in this study (n=12). All proceduresinvolving animals were approved by the Institutional Animal Care and UseCommittee at Northwestern University. The described research conformswith the Guide for the Care and Use of Laboratory Animals published bythe US National Institutes of Health (NIH Publication No. 85-23, revised1996). One week after instrumenting right ventricle pacing leads,open-chest baseline in vivo electrophysiology measurements were takenbefore gene vector delivery. After gene delivery, the chests wereclosed, and 3-5 days later, right ventricular tachypacing (240 bpm)commenced and was continued for 19 days to induce CHF.

Example 11 Gene Delivery

About 15 mg of Control(lacZ) or TbdnRII plasmid expression vector underthe control of the human polyubiquitin promoter (pUBc-lacZ (FIG. 10A) orpUBc-HA-TβdnRII (FIG. 10B)) was injected subepicardially in multiple(10-15) small volume (˜100 μL) injections dispersed across the posteriorleft atrium (PLA) followed immediately by electroporation (8 pulses at 1sec apart, amplitude 200 V, pulse duration for 10 ms), in accordance tothe method of Aistrup et al., Heart Rhythm. 2011 November; 8(11):1722-1729, the contents of which are hereby incorporated herein byreference in its entirety.

Example 12 AF Inducibility

In the initial study, AF duration was monitored for CHF recipients (FIG.11A and B). At the terminal study, AF was induced by burst atrialpacing. AF episodes (>30 s each) were recorded from the PLA and the LAAusing a high density mapping system with either Puka Cardiolab (GE), 7×3electrodes, 5 mm inter-electrode spacing; or Unemap (Univ of Auckland),130 electrodes, 2 mm inter-electrode spacing.

Example 13 Tissue Sample Preparation and Analyses

Immediately following the in vivo electrophysiology study, the heart waspromptly excised out of the chest and immersed in ice-cold cardioplegia.Tissue samples were taken from the PLA, pulmonary vein sleeve atria, andleft atrial appendage (LAA) regions of the left atrium and snap frozenin liquid nitrogen, and subsequently stored at −80° C. Some tissuesamples were homogenized and subsequently subjected to qRT-PCR forTβdnRII transcripts vs. endogenous TβRII (endogenous) transcripts; andanti-HA TβdnRII Western blot protein expression verification, andcontrol vs. TβdnRII PLA anti-TβRII Western blot analyses. Other tissuesamples were fixed/paraffin-embedded, sectioned (5 mm thick)transmurally from epicardium to endocardium, and either subjected toanti-HA-TβdnRII HRP colorimetric expression distribution analysis, orMasson's Trichrome staining and examined quantitatively for % fibrosis.

Example 14 Results

AF EGMs in HF Versus Normal Left Atrium

Dominant Frequency

Mixed effect ANOVA showed significantly lower mean DFs with HF than innormals (P=0.0002), but no significant difference in mean DF betweensites (P=0.65; FIG. 4A). Heterogeneity (SD) of DF was also lower in HFthan in normals, but with significant regional differences (that is,dispersion) within the left atrium (P=0.0007; FIG. 4B). SD of DF fornormals was significantly higher in PV than in the PLA and LAA (P<0.01),whereas SD of DF of the PV and PLA were significantly higher than theLAA with HF (P<0.02).

Organization Index

Mean OI was significantly higher in HF dogs than in normals (P=0.0001),with significant regional differences within the left atrium (P=0.0002;FIG. 4C). For normal dogs, the OIs were lower in the PLA than in the LAA(P<0.03). For HF dogs, the OIs were lower in the PV and PLA than in theLAA (P<0.04). SD of OI was not different between HF and normals (P=0.59)but showed regional differences within the left atrium (FIG. 4D). SD ofOI was significantly higher in the PLA than in the LAA (P<0.002).

Fractionation Interval

Mean FI was significantly higher in HF dogs than in normals (P<0.0001),with significant regional differences within the left atrium (P=0.003;FIG. 4E). For normal dogs, the FIs were significantly lower in the PVand PLA than in the LAA (P<0.03). SD of OI was significantly lower inthe HF dogs than in the normal dogs (P<0.0001) but showed no significantregional differences within the left atrium (FIG. 4F).

Percentage of CFAEs

Percentage CFAE was significantly lower in HF than in normals in the PV(72±4 versus 88±4%; P=0.002). PLA (59±4 versus 92±2%; P<0.001), and LAA(59±5 versus 80±6%; P=0.003). In HF, % CFAE was significantly greater inthe PV than in the PLA or LAA (P<0.05, for both comparisons). Innormals, % CFAEs were significantly greater in the PLA and PV than inthe LAA (P<0.05, for both comparisons).

Shannon Entropy

Mean ShEn trended lower in HF dogs than in normals (P<0.08), withsignificant regional differences within the left atrium (P=0.003; FIG.4G). For HF dogs, ShEn levels were significantly higher in the PV andPLA than that in the LAA (P<0.0006). SD of ShEn was not differentbetween HF dogs and normal dogs (P=0.14) or between sites (P=0.31; FIG.4H).

AF EGM Characteristics in the HF PLA Versus LAA (with High-DensityPlaques)

In both the PLA and LAA, there was no difference in DF between low- andhigh-density plaques (data not shown). However, OI was lower, FI wasgreater, and ShEn was lower with high-density plaques compared with thelow-density plaques (data not shown). This is likely because of thedifference in inter-electrode distance between the plaques; increasinginter-electrode distance for the same set of bipolar recordings resultsin a decrease in OI, decrease in FI, and increase in ShEn (data notshown). All the remaining AF mapping data below was obtained withhigh-density plaques. In the 1 dog that underwent both low- andhigh-density mapping, the differences between low- and high-densitymapping were consistent with the overall mean differences for all dogsbetween low-versus high-density mapping (data not shown).

Overall, differences between the PLA and LAA during high-density mapping(where the PLA and LAA were mapped sequentially) were similar to thosefound during low-density mapping (where the PV, PLA, and LAA were mappedsimultaneously). OI was significantly lower in the HF PLA compared withthe LAA, with ShEn trending toward being greater in the PLA than in theLAA (FIG. 5A). DF, OI, FI, and ShEn were all more heterogeneous in theHF PLA than the LAA (FIG. 5B).

Distribution of Fibrosis, Fat, and Nerves in the HF Left Atrium

The PLA had significantly more fat than the LAA (36.4±2.8% versus21.6±2.2%; P<0.001) (FIG. 6A, subpanel i.). Percentage myocardium wasgreater in the LAA than in the PLA (53.5±2.4% versus 35.6%±2.9%;P<0.001). There was no significant difference in fibrosis between thePLA and LAA.

Percentage fat was assessed in the PLA in a small number of normal dogs(n=3) and was found to be no different than in HF (36.4±2.8% versus30.1±2.1%; P=0.22).

Myocardium and fibrosis were more heterogeneously distributed in the PLAthan in the LAA (SD of % myocardium in PLA versus LAA=20.5±1.7% versus14.1±1.3%; P=0.01; SD of % fibrosis in PLA versus LAA=16.9±1.9% versus12.3±1.5%; P=0.02; FIG. 6A, subpanel ii.). Fat also trended toward beingmore heterogeneous in the PLA than in the LAA (17.3±1.7% versus12.3±2.3%; P=0.07).

FIG. 6B shows examples of significantly greater fat in the PLA(subpanels i. and iii.) than the LAA (subpanels ii. and iv.). Thesepanels also demonstrate that fat, fibrosis, and myocardium were moreheterogeneously distributed in the PLA than in the LAA. A significantnumber of nerve trunks were noted in the PLA fat (43±9; FIG. 6B,subpanel i., and FIG. 9C for examples of nerve trunks/bundles in thePLA). In contrast, no nerve trunks were found in the LAA.

Correlation Between AF EGM Characteristics and Fibrosis

DF was negatively correlated to % fibrosis (r=−0.45; P=0.006; FIG. 7A,subpanel i.), whereas FI was positively correlated with % fibrosis(r=0.42; P=0.01; FIG. 7A, subpanel ii.) in the PLA. Heterogeneity (SD)of DF and heterogeneity (SD) of FI were correlated with heterogeneity(SD) of fibrosis (for DF, r=0.41; P=0.01; for FI, r=0.47; P=0.004; FIG.7B, subpanels i. and ii., respectively).

FIG. 8A shows an example of OI being lower and more heterogeneous (thatis, greater SD) in the HF PLA than the LAA. FIG. 8B shows an example ofDF being more heterogeneous in the HF PLA compared with the LAA.Subpanels i. and ii. in each panel show the Masson-Trichrome stained PLAand LAA, respectively. Subpanels iii. and iv. in FIG. 8A show thecorresponding OI maps for each region mapped. Similarly, subpanels iii.and iv. in FIG. 8B show the corresponding DF maps for each regionmapped.

Effect of Double Autonomic Blockade on EGM Content in the HF Left Atrium

In the PLA, double autonomic blockade lead to a significant decrease inDF (from 6.8±0.6 to 6.1±0.7 Hz; P<0.001) and an increase in FI (from138±18 to 158±26 ms; P=−0.002; FIG. 9A). The increase in FI by doubleautonomic blockade was paralleled by a decrease in % CFAEs in the PLA(from 34±15% to 21±13%; P=0.01). A trend toward the decrease of ShEn wasnoted in PLA in the presence of double autonomic blockade (from0.76±0.01 to 0.73±0.01; P==0.098). No change in OI was noted in the PLAwith double autonomic blockade. In the LAA, there was no change in anyof these measures in the presence of double autonomic blockade (FIG.9A).

FIG. 9B shows examples of EGMs before and after autonomic blockade; asshown, the AF EGMs become significantly slower and less fractionatedafter autonomic blockade. FIG. 9C shows that with autonomic blockade, DFchanges significantly over regions of fat in the PLA. In FIG. 9C,subpanel i. shows the PLA being mapped. Subpanels ii. and iii. show theDF map before and after autonomic blockade; as shown, there is asignificant decrease in DF after autonomic blockade. Moreover, thedecrease in DF is most pronounced over regions of fat that contain largenerve trunks (encircled regions). Subpanel iv. highlights a large nervetrunk seen in subpanel i. In the PLA, change in ShEn (ΔShEn) withautonomic blockade was positively correlated with % fatty tissue(r=0.42; P<0.05; FIG. 7C).

Dominant Negative TGF-β R2 cDNA Gene Expression in Recipient PLA TissuesDecreases Fibrosis and AF in Ventricular-Tachypaced Canine Hearts.

A canine congestive heart failure (CHF) model was developed to testwhether administration of a dominant negative TGF-β R2 cDNA geneexpression vector (pUB6c-HA-TβdnRII) could decrease fibrosis and AF. TheAF characteristics of recipient subjects were monitored followinginjection into myocardial tissue (for example, PLA) of the controlplasmid (FIG. 10A) or the therapeutic DNA, pUB6c-HA-TβdnRII (FIG. 10B).Recipients that did not receive the therapeutic DNA displayed prolongedAF durations of about 600 seconds (FIG. 11A) and displayed an elevatedfrequency of prolonged AF episodes longer than 30 seconds upon induction(FIG. 11B). By contrast, recipients receiving the injection of thetherapeutic DNA displayed significantly lower AF durations (FIG. 11A)and at least ˜5-fold (or more) lower frequency of prolong AF episodeslonger than 30 seconds (FIG. 1B).

A molecular analysis of the PLA tissues from both cohort recipientgroups indicated that expression of TβdnRII mRNA was lower (˜33%) thanendogenous TβRII (FIG. 12) and that TβdnRII protein expression wasevident in TβdnRII cDNA injected recipients (FIG. 13A). Surprisingly,the amount of endogenous TβRII protein in PLA cells containing TβdnRIIprotein was 16% lower than the corresponding level of endogenous TβRIIprotein in PLA cells lacking TβdnRII protein (FIG. 13 B, C). The TβdnRIIprotein expression was nominally distributed homogeneously inpUBc-HA-TβdnRII-injected CHF PLAs, and was restricted to the CHF PLAs(FIG. 14). Notably, fibrosis was reduced in pUBc-HA-TβdnRII-injected CHFPLAs (FIG. 15C, D and FIG. 16C, D) vs. pUBc-lacZ-injected CHF PLAs (FIG.15A, B and FIG. 16A, B); see FIG. 16E (showing a significant reductionin % fibrosis for the CHF recipients injected with pUBc-HA-TβdnRIIcompared to pUBc-lacZ-injected recipients). These effects were stablymaintained and long-lived following injection of the pUBc-HA-TβdnRIIinto myocardia tissues (for example, evident for at least 24 days).

To the extent that the present application references a number ofdocuments, those references are hereby incorporated by reference hereinin their entirety.

While the present invention has been described with reference to certainembodiments, it will be understood by those skilled in the art thatvarious changes may be made and equivalents may be substituted withoutdeparting from the scope of the present invention. In addition, manymodifications may be made to adapt a particular situation or material tothe teachings of the present invention without departing from its scope.Therefore, it is intended that the present invention not be limited tothe particular embodiment disclosed, but that the present invention willinclude all embodiments falling within the scope of the appended claims.

The invention claimed is:
 1. A method of reducing AF fibrosis in asubject, comprising: (a) providing an isolated therapeutic DNAcomprising a dominant negative TGF-β R2 cDNA expression vector thatencodes and expresses dominant negative TGF-β R2 mRNA and protein invivo; (b) administering the isolated therapeutic DNA to myocardialtissue of the subject; (c) measuring a plurality of recorded atrial EGMsfor a region of myocardial tissue after administration of the isolatedtherapeutic DNA; and (d) continuing administration with the isolatedtherapeutic DNA based upon detecting an absence of one or more continuedsignificant changes in EGM characteristics in the plurality of recordedEGMs with administration of the therapeutic DNA; wherein the absence ofone or more continued significant changes in EGM characteristics in theplurality of recorded EGMs is indicative of a reduction in percentfibrosis not being retained at the region of myocardial tissue followingadministration of the isolated therapeutic DNA.
 2. The method of claim1, wherein step (c) comprises analyzing the plurality of recorded atrialEGMs using at least one analytical subroutine selected from the groupconsisting of dominant frequency analysis (DF), organizational indexanalysis (OI), fractional interval analysis (FI) and Shannon Entropyanalysis (ShEn).
 3. The method of claim 1, wherein the subject is apatient in need of preventative treatment for stroke or congestive heartfailure as a result of atrial fibrillation.
 4. The method of claim 1,wherein the myocardial tissue comprises PLA.
 5. The method of claim 1,wherein administering the isolated therapeutic DNA to myocardial tissueof the subject comprises injecting the isolated therapeutic DNA.
 6. Amethod of claim 1, further comprising assessing AF characteristicsfollowing administering the isolated therapeutic DNA.
 7. The method ofclaim 6, wherein the AF characteristics comprise at least one memberselected from the group consisting of AF duration and AF episodeinducibility.
 8. A method of reducing AF fibrosis in a subject,comprising: (a) providing an isolated therapeutic DNA comprising adominant negative TGF-β R2 cDNA expression vector that encodes andexpresses dominant negative TGF-β R2 mRNA and protein in vivo; (b)administering the isolated therapeutic DNA to myocardial tissue of thesubject; and (c) measuring a plurality of recorded atrial EGMs for aregion of the myocardial tissue before and after administration of theisolated therapeutic DNA to monitor the efficacy of the isolatedtherapeutic DNA.
 9. The method of claim 8, further comprising: (d)ceasing therapy with the isolated therapeutic DNA based upon detecting apresence of one or more continued significant changes in EGMcharacteristics in the plurality of recorded EGMs with administration ofthe therapeutic DNA; wherein the presence of one or more continuedsignificant changes in EGM characteristics in the plurality of recordedEGMs is indicative of a reduction in percent AF fibrosis being retainedat the region of myocardial tissue following administration of theisolated therapeutic DNA.
 10. The method of claim 8, further comprisingmeasuring the AF characteristics of the myocardial tissue, wherein theAF characteristics comprises at least one member selected from the groupconsisting of AF duration and AF episode inducibility.